Respiratory distress syndrome (RDS) is one of the most critical problems associated with the clinical management of premature newborns. In the United States, about 4,000 newborns die of RDS every year. RDS is a syndrome caused by a deficiency of pulmonary surfactant, a complex mixture of phospholipids and proteins necessary for proper pulmonary function. There would be great advantages to the accurate assessment of the lung maturity of a fetus at risk for premature delivery since in many cases birth can be delayed by clinical intervention. For premature infants born with immature lungs there are treatments available such as the administration of artificial surfactant. Thus, a convenient and accurate test for fetal and/or infant lung maturity would be very valuable in the clinical management and prevention of RDS.
Amniotic fluid samples containing fetal lung effluent ay be obtained through amniocentesis or from vaginal pooling. Samples of newborn infant lung fluid may be obtained through pulmonary lavage. These samples contain pulmonary surfactant in the form of lamellar bodies, which are essentially multilaminar lipid globules. Mature pulmonary surfactant is a complex mixture of lipids and proteins with less than 5% carbohydrates. Most of the lipid is phospholipid, and the majority of that, in mature lungs, is lecithin, also known as phosphatidylcholine. The structure of lecithin is shown below: ##STR1##
The R groups represent various fatty acid side chains. About 60% of the lecithin in mature surfactant has two palmitic acid side chains (dipalmitoyl lecithin).
Other lipids present in surfactant are phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and sphingomyelin. In general, the more mature the surfactant, the less viscous the lipid component thereof. Lecithin is less viscous than, for instance, sphingomyelin. As more mature surfactant is produced, the surfactant decreases in viscosity as the lecithin fraction rises in comparison to the sphingomyelin fraction. The structure of sphingomyelin is shown below: ##STR2##
The most widely accepted measure of lung maturity is the determination of lecithin/sphingomyelin ratio (L/S ratio) in samples of amniotic fluid or infant pulmonary effluent (Hamilton, P. R. et al., 1984, Obstetrics and Gynecology 153:52-56). Up to 34 weeks of gestation, the L/S ratio is approximately 1, after which there is a sudden rise in lecithin concentration (Gluck, L., et al., 1971, American J. Obstetrics and Gynecology 109:440-445). By week 35-36 of gestation, the L/S ratio reaches approximately 4. It is commonly accepted that an L/S value of 2 or greater is an accurate indication that the fetal lung is producing sufficient surfactant to prevent RDS at birth. Conversely, however, an L/S value of less than 2 is not an accurate predictor of fetal lung immaturity (Hamilton, P. R., et al supra). Several studies have shown that as many as 77% of fetuses with L/S less than 2 have not suffered RDS upon premature birth. Nevertheless, assessment of L/S ratio is considered valuable for the prediction of lung maturity since unnecessary clinical treatment may be avoided when the L/S value is greater than 2.
The L/S ratio may be determined by the direct measurement of lecithin and sphingomyelin, most commonly by thin layer chromatography (TLC) (Bustos, R., et al., 1979, Am. J. Obstet. Gynecol. 133:899). TLC is limited in its clinical usefulness because the technique is labor-intensive, lengthy, and requires a specially trained technician.
An indirect measure of L/S ratio is provided by the measurement of fluorescence polarization of chemical probes added to the sample of fetal lung effluent. One such chemical probe is the fluorescent dye DPH (1.6 diphenyl-1.3.5 hexatriene) (Shinitzky, M., et al., U.S. Pat. No. 4,071,770, issued Jan. 31, 1978; Blumenfeld, T. A., et al., Am. J. Obstet. Gynecol. 130:782-787). Another fluorescent dye used for this purpose is NBD-PC [(7-nitro-2,1,2-benzooxadiazol-4-yl)amino]caproyl phosphatidylcholine] (Russell, J. C., U.S. Pat. No. 4,784,961, issued Nov. 15, 1988). It is generally believed that these dyes integrate into the lung fluid lipids. The mobilities of these dyes are determined by the viscosity of the lipid phase which depends upon the ratio of the various lipids. The mobility of the dye is detected by the polarization of the fluorescence of the dye after illumination by the appropriate exciting light. In samples containing a relatively viscous lipid mix (i.e. low L/S ratio), the fluorescence polarization is relatively high. Lower fluorescence polarization values are correlated with decreased lipid viscosity and with higher L/S values. It should be noted that the term "fluorescence polarization" refers to conventional polarization which is dependent on the use and measurement of polarized light; this is distinguished from the property known as "generalized polarization" which does not depend on polarized light. A disadvantage of methods dependent on fluorescent probes such as DPH and NBD-PC is that they require the use of a delicate and expensive instrument capable of producing and measuring polarized light--a fluorescence polarization photometer.
One alternative method for the assessment of lung maturity is based on the capacity of amniotic fluid to form a stable foam in the presence of an alcohol (Statland, B. E., et al, U.S. Pat. No. 4,233,032, issued Nov. 11, 1980). Another method is based on the capacity of amniotic fluid to form bubbles in an ether layer (Socol, M., U.S. Pat. No. 4,547,464, issued Oct. 15, 1985).
In addition to correlations with L/S ratio, the presence in amniotic fluid of the lipid phosphatidylglycerol has been reported to correlate with fetal lung maturity. An assay has been patented which is based on the agglutination reaction caused by the addition of an antibody to phosphatidylglycerol (Yabusaki, K. K., U.S. Pat. No. 4,388,412).
Another test is based on a change in the optical properties of an amniotic fluid sample (Weitz, S. L., U.S. Pat. No. 4,820,628, issued Apr. 11, 1989). The optical change is caused by subjecting the sample to detergent or heat, which is thought to unravel or solubilize the lamellar bodies contained therein.
Although there exist many different methods for assessing lung maturity, the value thought to correlate best with fetal lung maturity remains the L/S ratio. There remains a clear need for a quick, accurate test for L/S ratio which can be easily performed in a clinical laboratory setting.